Methods for detecting and quantifying binding and inhibition of binding of species to nucleic acids

ABSTRACT

The invention features methods for detecting and quantifying the binding or inhibition of binding of species to biopolymers, e.g., nucleic acids. The invention is based on the use of probes that have magnetic relaxation properties that are affected by the presence of paramagnetic metal ions, e.g., Mn 2+ . Any class of biopolymer that binds metal ions at its active site or uses metal ion cofactors can be studied using these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. ProvisionalApplication No. 60/332,969, filed Nov. 6, 2001, hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The invention relates to the fields of drug discovery and nuclearmagnetic resonance spectroscopy.

Nucleic acids (DNA and RNA) are biopolymers in which each monomercontains a negatively charged phosphate ester group. This large negativecharge is balanced by interaction of the nucleic acid with smallcationic species, including protonated amines, and monovalent anddivalent metal ions. In addition to non-specific electrostaticinteractions, structured nucleic acids have specific metal ion bindingsites. Examples of specific divalent metal ion binding by RNA moleculescan be seen in the group III-V intron of the tetrahymina ribozyme,ribosomal RNA, and RNA aptamers. Such interactions may be mediated bydirect coordination of the bases or phosphates to the metal ion, or byhydrogen bonding between water molecules coordinated to the metal ionand the nucleotide bases.

Divalent metal ions can be bound by either specific interactions withdiscrete metal binding sites or non-specifically via coulombicinteraction. For non-specific interactions, binding is described by thepolyelectrolyte condensation model. Binding affinities are dependant onthe ionic strength of the solution and the nature of the cationic formof the supporting electrolyte. This dependence on the nature andconcentration of the supporting electrolyte is to be expected since thedivalent ion will have to compete with the electrolyte cation for bothspecific and non-specific interactions. Non-specific affinities can berepresented by interactions of Mg²⁺ with poly-uridine (poly-U). In 10 mMNaCl, 96 μM Mg²⁺, the apparent association constant for poly-U/Mgbinding is reported as 1.86×10³ M⁻¹nt⁻¹, where nt is the number ofnucleotides in the strand. When other bases are incorporated into theRNA strand, binding of the metal to the bases is possible. Under similarconditions to those of the poly-U study, poly-A has a reported Mg²⁺binding affinity of 10.7×10³ M⁻¹nt⁻¹, a 5.7 fold increase over poly-U.This difference must be attributed to contributions from base/metalinteractions, since the two nucleic acids share the same chargedistribution along their backbones. Specific metal binding sites havingaffinities in the micromolar range have been reported for transfer RNAsand the hammerhead ribozyme. Up to 8 Mn²⁺ ions bind to the Hammerheadribozyme with K_(D) values ranging from 4 to 500 μM.

Since divalent metal ions are necessary structural elements of somemolecules of RNA and since the RNA molecules exchange bound ions withions free in the bulk solution, sites for binding metal ions are naturalcandidates for drug targets. Aminoglycoside antibiotics are believed toact by displacing divalent metal ions from specific binding sites onRNA. Aminoglycosides are reported to displace Mg²⁺ from specific siteson a variety of RNA molecules including a model of the ribosomal A, thehammerhead ribozyme, the tetrahymina group I intron, and RNase P.

The magnetic relaxation properties of NMR active nuclei can be verysensitive to paramagnetic metal ions. Thermodynamic and structuralaspects of macromolecules that bind metal ions have been studied byexploiting this effect. In the majority of works, the effects of theparamagnetic metal ion on the magnetic resonances of ¹H of thebiological molecule have been determined in order to study the geometryof the metal binding site. In addition, there have been a more limitednumber of studies where the metal binding sites have been examined usingexternal small species such as water or fluoride ion.

Paramagnetic relaxation enhancement of the magnetic resonance of ¹Hnuclei of solvent water has been used to study metal binding by avariety of biological macromolecules qualitatively. This approach hasbeen used to characterize the binding of Mn²⁺ to transfer RNA andribosomal RNA among other molecules. Another approach for paramagneticrelaxation enhancement that has seen limited use is the use of ¹⁹Ffluoride as a probe. Relaxation enhancements of this resonance bysuperoxide dismutases have been reported. Metal ion binding studieswhere the perturbations observed are of the macromolecular ¹H resonanceshave also been reported. Molecules studied include ribozymes and smallerRNA fragments. Water ¹H resonances, however are not very sensitive toparamagnetic relaxation, and ¹⁹F measurements require ¹⁹F NMR.

There exists a need for sensitive methods for measuring the binding andinhibition of binding of species to nucleic acids. These methods mayfind use in the field of drug discovery.

SUMMARY OF THE INVENTION

The invention provides methods for detecting and quantifying the bindingor inhibition of binding of species, e.g., candidate therapeutic agents,to biopolymers, e.g., nucleic acids. The invention is based on the useof probes that have magnetic relaxation properties that are affected bythe presence of paramagnetic metal ions. Although the followingdescription focuses on nucleic acids, any class of biopolymer that bindsmetal ions at its active site or uses metal ion cofactors can be studiedusing these methods.

In one aspect, the invention features a method of detecting the bindingof a species to a nucleic acid that includes the steps of measuring amagnetic relaxation property of a probe in a first solution usingnuclear magnetic resonance spectroscopy, wherein the first solutionincludes the nucleic acid, a first concentration of the species, aparamagnetic metal ion, and the probe; measuring the magnetic relaxationproperty of the probe in a second solution using nuclear magneticresonance spectroscopy, wherein the second solution includes the nucleicacid, a second concentration of the species, a paramagnetic metal ion,and the probe; and comparing the relaxation properties measured, whereina difference in the relaxation properties indicates the binding of thespecies to the nucleic acid. In one embodiment, the magnetic relaxationproperties of the probe are correlated with the first and secondconcentrations of the species, thereby quantifying the binding of thespecies to the nucleic acid.

The invention further features a method of detecting the binding of aparamagnetic metal ion to a nucleic acid including the steps ofmeasuring a magnetic relaxation property of a probe in a first solutionusing nuclear magnetic resonance spectroscopy, wherein the firstsolution includes a first concentration of the paramagnetic metal ion,the nucleic acid, and the probe; measuring the magnetic relaxationproperty of the probe in a second solution using nuclear magneticresonance spectroscopy, wherein the second solution includes a secondconcentration of the paramagnetic metal ion, the nucleic acid, and theprobe; and comparing the relaxation properties measured, wherein adifference in the relaxation properties indicates the binding of theparamagnetic metal ion to the nucleic acid. In one embodiment, themagnetic relaxation properties of the probe are correlated with thefirst and second concentrations of the nucleic acid, thereby quantifyingthe binding of the paramagnetic metal ion to the nucleic acid.

In another aspect, the invention features a method of determining theeffect of a first species on the binding of a second species to anucleic acid including the steps of measuring a magnetic relaxationproperty of a probe in a first solution using nuclear magnetic resonancespectroscopy, wherein the first solution includes a first concentrationof the first species, the nucleic acid, the second species that binds tothe nucleic acid, a paramagnetic metal ion, and a probe; measuring themagnetic relaxation property of the probe in a second solution usingnuclear magnetic resonance spectroscopy, wherein the second solutionincludes a second concentration of the first species, the nucleic acid,the second species, the paramagnetic metal ion, and the probe; andcomparing the relaxation properties measured, wherein a difference inthe relaxation properties indicates the inhibition of the binding of thesecond species to the nucleic acid. In one embodiment, the magneticrelaxation properties of the probe are correlated with the first andsecond concentrations of the first species, thereby quantifying theeffect of the first species on the binding of the second species to thenucleic acid.

The invention also features a method of detecting the availability of aparamagnetic metal ion in a solution comprising a nucleic acid includingthe steps of providing a solution comprising the nucleic acid, a probe,and the paramagnetic metal ion; and measuring a magnetic relaxationproperty of the probe in the solution using nuclear magnetic resonancespectroscopy, wherein the magnitude of the magnetic property isindicative of the availability of the paramagnetic ion in the solution.

In various embodiments of the above aspects, the first (or second)concentration of a species or ion may be 0.0 μM. In addition, a secondconcentration may be lower or higher than a first concentration, and asecond solution may be formed by the addition or removal of species orions from the first solution. Exemplary species include metal ions(e.g., Mg²⁺) and candidate therapeutic agents (e.g., proteins, peptides,or fragments thereof). Desirably a candidate therapeutic agent isselected from a molecular library (e.g., a combinatorial library). Thenucleic acid may be RNA or DNA and may also be double-stranded or singlestranded. Exemplary paramagnetic metal ion include, without limitation,Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, and a lanthanide ion. In otherembodiments, the paramagnetic ion is bound to the nucleic acid.Desirably, the probe includes an X—H bond, wherein X is an NMR-activenucleus. In desirable embodiments, X is ³¹p, such as in methylphosphiteor ethylphosphite. The probe may also be an alkylphosphonite, e.g.,ethylphosphonite.

In addition, the magnetic relaxation properties of X may be indirectlydetected using X edited, ¹H detected NMR spectroscopy. In variousembodiments, the magnetic relaxation property is the T₂ relaxation of anucleus of the probe. Desirably, the measuring of said magneticrelaxation properties of X proceeds by the steps of using a pulsesequence to transfer coherent magnetization originating on the ¹Hnucleus to the X nucleus by Insensitive Nuclei Enhanced by PolarizationTransfer (INEPT) techniques prior to a T₂ delay; providing a T₂ delay;and transferring the remaining coherence back to the ¹H nucleus fordetection using a reverse INEPT series of pulses.

In another aspect, the invention features a kit for screening forspecies that bind to or inhibit binding to nucleic acids including anucleic acid, a paramagnetic metal ion, and a probe, as describedherein. The kit may further include an NMR spectrometer.

By “probe” is meant any molecule or species having at least one NMRactive nucleus that undergoes a significant increase in transverserelaxation time (T₂) when in a solution containing a paramagnetic metalion. Examples of NMR-active nuclei include, but are not limited to, ¹H,¹³C, ³¹P, ⁵N, ¹⁷O, and ¹⁹F (see Yoder, C. H., et al. Introduction toMultinuclear NMR, Cummings:Menlo Park 1987, pp 317–319). Desirableprobes include those that contain X—H bonds, where X is a non-hydrogenNMR-active nucleus. More desirably the probes contain P—H bonds.Particularly desirable probes are methylphosphite and ethylphosphonite.Other suitable probes contain only one NMR-active nucleus of an elementother than hydrogen, e.g., ¹⁵N or ³¹P.

By “species” is meant a molecule, atom, radical, ion, or metal ion.Desirable species include, but are not limited to, members of molecularlibraries, inorganic compounds, synthetic molecules, natural products,antibiotics, drugs, drug candidates, derivatives of natural products,proteins, peptides, fragments of proteins, and Mg²⁺.

By “nucleic acid” is meant substituted or unsubstituted RNA or DNA.Desirable nucleic acids are RNA molecules.

By “NMR-active nucleus” is meant a nucleus having a non-zero net nuclearspin.

By “biopolymer” or “biological polymer” is meant a protein, polypeptide,nucleic acid, or polysaccharide. The biopolymer may include naturallyoccurring components or synthetically modified components. In desirableembodiments, the biopolymer is a nucleic acid.

By “candidate therapeutic agent” is meant a chemical species that isidentified by an assay as potentially having therapeutic efficacy for aspecified condition. Exemplary candidate therapeutic agents include,without limitaion, natural and synthetic organic compounds, natural orsynthetic inorganic compounds, natural or synthetic peptides orpolypeptides, and nucleic acids.

By “bound to the nucleic acid” is meant coordinated to the nucleic acidand not free in solution.

By “availability” of species in solution is meant the ability of a probeto interact with the species to an extent that a magnetic property ofthe probe is altered. Species free in solution have a greateravailability than those coordinated to a nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic representations of chemical structures ofmethylphosphite (MeOPH) and ethylphosphonite (EtPH), respectively.

FIG. 2 is a schematic representation of one-dimensional pulse sequencesfor measuring ³¹P T₂ values using ¹H detection. Broad and narrow barsrepresent 180- and 90-degree pulses, respectively.

FIG. 3 is a graph showing the intensity (i) of the ¹H signal ofethylphosphite (EtOPH) obtained using the pulse sequence of FIG. 2 as afunction of the length of the CPMG pulse train.

FIG. 4 is a graph showing the inverse of the T₂ relaxation time (1/T₂)of EtOPH ³¹P with and without RNA present as a function of theconcentration of Mn²⁺.

FIG. 5 is a graph showing the inverse of the T₂ relaxation time (1/T₂)of EtOPH ³¹P in the presence of 5.3 μM Mn²⁺ as a function of RNAconcentration.

FIG. 6 is a graph showing the concentration of free Mn²⁺ in the presenceof 3.6 μM of RNA as a function of the concentration of the nucleic acidbinding compound MES 10094.

FIG. 7 is a graph showing the effect of RNA concentration onparamagnetic T₂ relaxation enhancement by Mn²⁺.

FIG. 8 is a graph showing the effect of a 27-nt A-site RNA construct onprobe ion relaxation enhancement by Mn²⁺.

FIG. 9 is a graph showing the displacement of bound Mn²⁺ from a 27-ntA-site RNA construct by neomycin.

FIG. 10 is a graph showing the effect of electrolyte concentration onrelaxation enhancement, expressed as a fraction of enhancement expectedin the absence of RNA, in solutions of a 27-nt A-site RNA construct andMn²⁺.

FIG. 11 is a graph showing Mn²⁺ binding by a 27-nt A-site RNA constructunder different salt concentrations.

FIG. 12 is a schematic description of a one dimensional pulse sequencefor measuring phosphite ³¹P T₂ values. Coherence originating on thephosphorus bound proton (¹H—P) nucleus is transferred to the ³¹P nucleususing a series of inept pulses. After a series of CPMG 180 degreerefocusing pulses (³¹P frequency), the remaining signal is returned tothe ¹H—P nucleus for detection using a second series of inept pulses.The transverse relaxation times (T₂) were determined from the slopes ofthe linear plots of log(i) versus t where i is the maximum intensity ofthe resonance, and t is the time between the two inept series of pulses(t=4nδ). Narrow and broad lines represent 90 and 180 degree pulses onthe indicated channel. A 90 degree ³¹P pulse and two homospoil pulsesare inserted into delay 1 to remove artifacts arising from residualmagnetization remaining between transients. All pulse phases are zerounless noted otherwise. Delays in the inept 1 and inept 2 portion of thesequence (Δ) are equal to 1/4J. The delay between CPMG pulses (δ) was 4ms. The T₂ delay was varied by changing the number of times through theCPMG cycle (n). Phase cycles were as follows: a=(1,1,1,1,3,3,3,3),b=(2), c=(3,3,3,3,3,3,3,3,1,1,1,1,1,1,1,1), d=(0,2), e=(0,0,2,2), andobs=(2,0,0,2,0,2,2,0).

FIG. 13A is a schematic depiction of RRE1, RRE2, and A-site hairpinstructures.

FIG. 13B is a schematic depiction of neomycin b.

FIG. 13C is a schematic depiction of RSG1.2.

FIG. 14 is a semi-log plot showing the effects of [Mn²⁺] on ³¹Ptransverse relaxation rates (R_(2,obs)) of phosphite dianion (HPO₃ ²⁻,represented by the X within the error bars) and methyl phosphite anion(CH₃OP(H)O₂ ⁻, represented by a +), corrected for diamagnetic relaxationrate (R_(2,dia)). R₂ of HPO₃ ²⁻ is a factor of ten more sensitive to[Mn²⁺] than the R₂ of MeOPH. Lines show behavior predicted forrelaxation enhancement proportional to [Mn²⁺].

FIG. 15 is a graph of the effect of A-site RNA on free Mn²⁺ (5 mM PIPES,pH 7.2, 5 mM MeOPH, K⁺ salts). The line represents behavior predictedfor 7 independent binding sites having K_(d,Mn)=25 μM.

FIG. 16 is a graph of the effect of [K⁺] on free Mn²⁺ in solutions ofA-site (27 μM) and Mn²⁺ (16 μM), as determined from R₂ data (FIG. 14).

FIG. 17 is a graph of the effect of [Mg²⁺] on MeOPH relaxation insolutions of RRE1 (20 μM) in 25 mM K⁺ (filled triangles) or 100 mM K⁺(open diamonds) and Mn²⁺ (3 μM). Solid lines show behavior predicted bythe K_(d) and n values in Table 1.

FIG. 18 is a graph of the effect of neomycin b on the relaxation rate(R₂) of the HPO₃ ²⁻ ³¹P signal in solutions of A-site RNA (2.7 uM), Mn²⁺(0.11 uM), and 100 mM K⁺.

FIG. 19 is a graph of the effect of RSG1.2 on Mn²⁺ binding by either 2.7μM RRE1 (filled triangles) or 3.0 μM RRE2 (open diamonds) in 100 mM K⁺.Values were normalized to the R₂ value, obtained upon addition of 2 mMMg²⁺

FIG. 20 is a graph of the effect of [RRE1] (filled triangles) and [RRE2](open diamonds) on fluorescence polarization of TAMRA labeled RSG1.2.

DETAILED DESCRIPTION OF THE INVENTION

The invention features methods for detecting binding or inhibition ofbinding of a species to a biopolymer, e.g., a nucleic acid. Certainchemical species can be used as probes for studying metal coordinationby such a biopolymer. The presence of paramagnetic metal ions affectsthe magnetic relaxation properties of nuclei in these probes asmeasured, for example, by NMR. The accessibility of paramagnetic metalions to the probe in solution correlates with the magnitude of theseeffects on magnetic relaxation.

Certain biopolymers bind metal ions in solution. The addition or removalof species that affect the quantity of metal ions bound to a biopolymer,e.g., a nucleic acid, can be studied using a suitable probe. Methodsbased on this phenomenon can be used to study, for example, binding ofmetal ions directly to nucleic acids, species that bind to nucleic acidsand displace metal ions, and species that inhibit the binding of otherspecies to nucleic acids. The methods may also be used to study speciesthat expose or conceal metal ions bound to biopolymers to allow orprevent interaction with a probe species, respectively.

Paramagnetic Metal Ions

Metal ions bound in vivo to biopolymers can usually be replaced withparamagnetic metal ions. For example, magnesium ions (Mg²⁺) are normallyfound in vivo in nucleic acid complexes and can usually be replacedwith, for example, manganous ion (Mn²⁺). Ions such as Mn²⁺ often supportthe structural and reactivity requirements of nucleic acids and theircomplexes for processing and binding proteins. The chemistry of Mn²⁺also closely approximates that of Mg²⁺. These two cations show similarpreferences for six coordination sites and have similar bond energiesand Lewis acidities. While the chemistry of Mn²⁺ and Mg²⁺ are similar,their magnetic properties are very different. Mg²⁺ is diamagnetic(having no unpaired electrons), and Mn²⁺ is paramagnetic (containingfive unpaired electrons). Additional exemplary paramagnetic ions thatare useful in the methods described herein include, without limitation,Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, or a lanthanide ion.

Probes

Magnetic resonances of certain nuclei can be highly sensitive toparamagnetic ions, and compounds containing these NMR-active nuclei canbe used as probes for the presence of paramagnetic metal ions. Forexample, the ³¹P nuclei of certain anionic materials includingmethylphosphite are remarkably sensitive to magnetic T₂ relaxation byMn(H₂O)₆ ²⁺ (or free Mn²⁺). Changes in the ³¹P NMR transverse relaxationrates of MeOPH can be used, for example, to detect and quantify theconcentration of Mn²⁺ in aqueous solutions at concentrations below 1 μM.

Previous work has measured magnetic relaxation of molecules in closeproximity to paramagnetic metal ions, but significant differences existbetween the methods of this invention and those of the prior art. Inprevious approaches, it is the magnetic resonances of the biopolymers,e.g., nucleic acids, themselves that are observed. Methods that centeron measuring the effect of metal ions have on the resonances of thebiopolymers are unlikely to develop into reasonable screens for drugactivity. Such a method would require that the biopolymers be present inthe solutions at concentrations of hundreds of μM or above. Since theanalyte must also be present at this concentration, the effects of weakinteractions (K_(d˜)100 μM) cannot be distinguished from those ofstronger interactions. Since compounds that only weakly interact areunlikely to make good drug candidates, and weak binders cannot bedifferentiated from stronger binders, these methods are of limited use.The concentration limitations also mean that drugs with lowersolubilities cannot be studied by these methods. Since the methods ofthe present invention observe the resonances of a probe, they are notlimited to working with high concentrations of biological species andcan be used to screen for drug hits at more physiological conditions.

Another limitation on methods where macromolecular resonances areobserved arises from the spectral overlap of these resonances:resolution and assignment of these resonances would requiremulti-dimensional spectroscopy of isotopically labeled samples. Anadditional benefit arises for the methods described herein since they donot require the assignment of the macromolecular resonance chemicalshifts. Macromolecules have many nuclei of a given element, e.g., H, C,N, or P, that may be present in chemical environments that only slightlydiffer. Since small differences in chemical environment give rise tosmall differences in chemical shift, the precise assignment of onenucleus out of the many similar nuclei is difficult.

The probes described herein form a complex with the paramagnetic metalions. This complex is weakly bound relative to complexes of the metalions and nucleic acids. Complexation of the probe to the metal ionaffects the magnetic relaxation properties of the probe. To achieve themaximum sensitivity in methods that measure metal ion availability insolution, interactions between the probe are paramagnetic metal ion aredesirable minimized when the ion is bound to a biopolymer. For example,one method to minimize the interaction of the probe with metal ionsbound to nucleic acids is to make the probe anionic. Since the metal ionis sequestered by a poly-anionic nucleic acid, the overall charge willbe highly negative and repel the anionic probe ion. Thus, changes in themagnetic relaxation properties by the sequestered metal ion are notobserved. If association with monovalent cations neutralizes the anioniccharge of the nucleic acid, then relaxation enhancement by the nucleicacid/Mn²⁺ complex is possible. In methods that measure the accessibilityof a metal ion bound to a biopolymer, however, the probe desirablyinteracts with the bound metal. For example, the probe may be of theopposite charge to the biopolymer, or it may be neutral.

Exemplary probes are shown in FIG. 1. These probes have at least one P—Hmoiety and at least one terminal P—O moiety, examples include alkylphosphite anions (e.g., methylphosphite, MeOPH, and ethylphosphite,EtOPH) and phosphonite anions (e.g., ethylphosphonite, EtPH). Theseprobe ions are far more sensitive to metal ions than the water ¹Hresonance and have the advantage over fluoride of allowing ¹H detectionwith isotope editing (Dwek, R. A. Nuclear Magnetic Resonance inBiochemistry Clarendon:Oxford 1973).

NMR

Nuclear magnetic resonance (NMR) spectroscopy is a widely used techniqueto study chemical and structural properties of species. The ability todetect magnetic relaxation rates, e.g., from T₁ and T₂ relaxations,provides a route to probe the environment of a particular nucleus.

For probes containing X—H bonds, the T₂ relaxation of the X nucleus canbe monitored using ¹H detection and isotope editing. For example, apulse sequence for a one-dimensional NMR experiment used to determinechanges in ³¹P T₂ of phosphites or phosphonites with indirect detectionis shown in FIG. 2. In this example, coherent magnetization originatingon the ¹H is transferred to the ³¹P via inept sequence (a). The sampleis pulsed with a series of 180-degree phosphorus re-focusing pulses(during the CPMG train), and magnetization is transferred back to the ¹Hnucleus for detection (inept sequence (b)). Delays between pulses in thetwo inept sequences are set to 1/4J. Pulse phases are those described byFarrow et al., for a ¹⁵N T₂ correlation experiment (Farrow, N. A. et al.Biochemistry, 1994, 33:5984–6003). The number of 180-degree phosphorusrefocusing pulses during the CPMG portion of the sequence and the delaybetween these pulses can be adjusted to alter the total time in the CPMGtrain. During this time (which is called the T₂ time) transverserelaxation of the ³¹P nucleus occurs, leading to a diminution of theintensity (i) of the observed signal. For example, the effect ofvariations in T₂ time on the intensity of the phosphite ¹H signal ofEtOPH in a solution containing 5 μM Mn²⁺ is presented in FIG. 3. In thisexample, the delay between ³¹P 180-degree pulses was held fixed at 200μs, and the number of pulses increased as the length of the CPMG pulsetrain increased. For example, the shortest time corresponds to 8 CPMGpulses, and the longest time corresponds to 160 pulses. The transverserelaxation time of the ³¹P nucleus of the probe under these conditionsis equal to the magnitude of the slope of the plot. While thisparticular example is for ³¹P nuclei, the technique is generallyapplicable to other NMR-active nuclei X that form strong X—H bonds.

The sensitivity of the technique depends, e.g., on the reproducibilityof the instrumentation, sample variability, and any paramagneticimpurities or dissolved oxygen present in the sample. The sensitivityalso depends strongly on the charge of the biopolymer. For example forRNA in low salt solutions, changes in Mn²⁺ concentration of ˜300 nM aredetected with suitable reproducibility.

Applications

The methods of the present invention can be used to study the relativeor absolute increase or decrease in the accessibility of metal ions insolution.

The ability to detect and quantify the concentration of freeparamagnetic metal ions in solution provides methods, for example, toscreen a series of drug candidates for species that bind to biopolymers,e.g., a nucleic acid, or inhibit the binding of other species tobiopolymers, to determine the stoichiometry of metal ions binding tobiopolymers, or to determine relative or absolute affinities of speciesbinding to biopolymers. The ability to detect and quantify theaccessibility of a metal ion bound to a biopolymer provides methods, forexample, to screen for ligands that bind or inhibit the binding ofspecies to the metal or biopolymer.

In one embodiment, species that bind to nucleic acids displace boundparamagnetic metal ions and cause an increase in the concentration ofparamagnetic metal ions free in solution. This increase in concentrationis measured, for example, by NMR as an increase in the T₂ relaxationrate of nuclei in a probe. The T₂ relaxation rates are then correlatedwith the concentration of species that binds to nucleic acid to detector quantify the amount of paramagnetic metal ions freed upon binding ofthe species to nucleic acid. The species that binds the nucleic acid maybe, e.g., a synthetic organic molecule, a peptide, or a protein.

In another embodiment, nucleic acid is added to a solution of freeparamagnetic metal ions. The ions then bind to the nucleic acid. Thisbinding is measured, for example, by NMR as a decrease in the T₂relaxation rate of nuclei in a probe. The change in the rate ofrelaxation of nuclei in the probe is then correlated with theconcentration of nucleic acid to detect or quantify the binding ofparamagnetic metal ions to the nucleic acid.

In still another embodiment, a species that potentially inhibits thebinding of other species, e.g., a protein, to nucleic acids is added toa solution containing a species bound to a nucleic acid and paramagneticmetal ions. Desirable proteins for binding inhibition include, withoutlimitation, the Hu antigens and AUF1 (Wilson, G. M. et al. J. Biol.Chem. 2001, 276:8695–8704). The inhibiting species reduces theconcentration of the other species bound to the nucleic acid, whichallows paramagnetic metal ions to bind to the nucleic acid. The bindingof paramagnetic metal ions to the nucleic acid reduces the concentrationfree in solution. The inhibition of binding of the species to thenucleic acid is measured, for example, by NMR as a decrease in the T₂relaxation rate of nuclei in a probe caused by the reduced concentrationof metal ions in solution. The change in the rate of relaxation of thenuclei in the probe is then correlated with the concentration of theinhibiting species to detect or quantify the inhibition of speciesbinding to nucleic acid.

EXAMPLES

The following examples are provided merely to illustrate variousfeatures and other details of the invention and should not be construedas limiting.

In Examples 1–15, the effects of RNA on equilibrium concentrations ofMn²⁺ are determined using a phosphite probe ion (either Methylphosphite, MeOPH, or Ethyl phosphite, EtOPH). Typically, samples ofMeOPH were prepared at (5–10 mM) in PIPES buffer (5–10 mM), which isknown not to interfere with metal ion availability (Kandegedara A. etal. Anal. Chem. 1999, 71:3140:3144). Aliquots of stock solutions wereevaporated in vacuo and reconstituted in D₂O (all experiments used D₂Oas solvent). Samples were treated with aliquots of Mn²⁺ in the presenceand in the absence of the RNA of interest.

Example 1 Synthesis of MeOPH and EtOPH

MeOPH was prepared by partial hydrolysis of dimethyl phosphite inaqueous base. Dimethyl phosphite (for MeOPH) or diethyl phosphite (forEtOPH) (Aldrich) was dissolved in deionized water, and the resultingsolution was neutralized by addition of an equivalent of either sodiumhydroxide (to prepare the sodium salt) or potassium hydroxide (toprepare the potassium salt). The progress of the reaction was monitoredusing a pH meter. The rate of addition was such that the temperature ofthe solution was not allowed to exceed ˜60° C. On a gram scale (˜2 Mconcentration), the process is complete in ˜5 minutes. Aliquots of thesample were then evaporated in vacuo to give a colorless powder thatcould be dissolved into D₂O for spectroscopic studies. The phosphorusbound proton did not exchange readily with those of the solvent.

Example 2 Synthesis of EtPH

EtPH was prepared by hydrolysis of ethyl-dichloro-phosphine. To a beakerof deionized water in an ice bath was added the contents of a one-gramampoule of ethyl-dichloro-phosphine with stirring. A vigorous exothermicreaction ensued. The sample was neutralized with aqueous base, andaliquots were evaporated in vacuo.

Example 3 Measurement of T₂ Relaxation Rates with NMR Spectroscopy

The ³¹P T₂ was measured for each sample by varying the length (number ofpulses) of the CPMG portion of the pulse sequence in FIG. 2 anddetermining the effect of this change on the intensity (i) of thesignals. Plots of ln(i) versus time (as seen in FIG. 3) were linear witha slope equal to the negative of the ³¹P T₂. The dl delay was typically5 to 20 seconds, and delays in the inept and reverse inept parts of thesequence were set to a value equal to 1/4J, where J is the ¹H—³¹Pcoupling constant. Delays between pulses in the CPMG train varied from50 to 500 microseconds.

Example 4 Mn²⁺ Binding Properties of a 38 Nucleotide RNA

A TNF-α mRNA construct binds divalent metal ions in a way that poly-Udoes not (Wilson, G. M. et al. J. Biol. Chem. 2001, 276:8695–8704).Complex formation between the RNA binding protein AUF1 and its targetRNA is inhibited by Mg²⁺, and RNA binds Ca²⁺ and Mn²⁺ as well as Mg²⁺.These results indicate that metal ions are released from specific siteson RNA when bound to AUF1.

A solution of EtOPH (5 mM) in D₂O was prepared and aliquots were treatedwith aliquots of Mn²⁺. Similar samples were prepared but with a 38 ntARE RNA construct at a concentration of 200 μM. The sequence of this RNAwas identical to that reported (Wilson, G. M. et al. J. Biol. Chem.2001, 276:8695–8704) to bind divalent metal ions. In the absence of theRNA, the measured value of the ³¹P T₂, obtained as described in Example3, decreased with the addition of metal ion. When the reciprocal of T₂of the ³¹P nucleus was plotted versus Mn²⁺ concentration, a linearcorrelation was obtained (open diamonds in FIG. 4). In contrast, the ³¹PT₂ of the probe was not measurably affected by the same level of Mn²⁺ inthe presence of the RNA (filled squares in FIG. 4). These resultsindicated that Mn²⁺ was effectively sequestered by the RNA and that,once sequestered, Mn²⁺ ions did not affect the relaxation properties ofthe probe ion.

Example 5 Determining the Affinity of the RNA for Mn²⁺

Example 4 demonstrated that the affinity of the 38 nt ARE construct forMn²⁺ was sufficient to reduce the level of free Mn²⁺ from 5.3 to lessthan 1 μM. To estimate the binding affinity of the nucleic acid forMn²⁺, samples containing the probe ion (EtOPH, 5 mM) and Mn²⁺ (5.3 μM)were treated with aliquots of the RNA construct (final concentrations of0, 1.4, 2.9, 4, 6, 10, 14, 58, and 232 μM), and the values of the probeion ³¹P T₂ were measured for each sample as in Example 3. A plot ofreciprocal T₂ versus RNA concentration (FIG. 5) shows that metal ionsequestration was efficient for this sequence well below 10 μM. Thisfigure also shows that more than one metal ion was bound per molecule ofnucleic acid under these conditions.

Example 6 Determining the Affinity of a Drug Candidate for RNA

To determine the affinity of an RNA-binding drug candidate, MES10094,disclosed in U.S. application Ser. No. 10/117,955 filed on Apr. 8, 2002,for the 38 nt RNA, a sample containing Mn²⁺ (5.3 μM), probe (5 mM), andRNA (3.6 μM) was treated with a concentrated solution of MES 10094. Theeffect of increasing concentration of the drug lead on the ³¹P T₂,measured as in Example 3, is presented in FIG. 6. This figure clearlyshows that addition of MES 10094 resulted in a pronounced increase inthe rate of probe ion ³¹P T₂ relaxation. This result was consistent withthe drug candidate displacing a fraction of the bound Mn²⁺ from the RNA.The data in FIG. 6 indicated that the K_(D) for metal displacement byMES 10094 under these conditions was on the order of 30 μM, which wasconsistent with values for inhibition of AUF1 binding to the RNA by MES10094.

Example 7 Determining the Affinity of a Drug Candidate for a Speciesthat Binds to RNA

To determine the affinity of a drug candidate for an RNA-bindingspecies, e.g., a protein, a sample containing Mn²⁺ (5.3 μM), probe (5mM), RNA-binding species (over a concentration range that spans an orderof magnitude greater than and less than the expected K_(d)), and RNA(3.6 μM) is treated with a drug candidate. The effect of increasingconcentration of the drug lead on the ³¹P T₂, measured as in Example 3,is then determined. The addition of a successful drug lead results in apronounced decrease in the rate of probe ion ³¹P T₂ relaxation. Thisresult is consistent with the drug candidate preventing the binding ofthe RNA-binding species and allowing a fraction of the free Mn²⁺ to bindto the RNA. The K_(D) for binding to the RNA-binding species under theseconditions is determined from curve fitting the data.

Example 8 Screening a Molecular Library for Drug Candidates

Species in a molecular library are screened to determine drug candidatesthat bind to RNA or inhibit binding of other species to RNA. Screeningfor RNA-binding candidates proceeds as in Example 6, and screening forcandidates that inhibit binding of other species to RNA proceeds as inExample 7. Several compounds may be screened at once. If a mixturecontains a drug candidate, the individual species are then tested todetermine the active species.

Example 9 Determining Non-Sequence Specific Association of Metals Ionswith RNA

To establish the effects of non-specific interactions, metal ionassociation with a 31-nucleotide poly-U RNA (U₃₁) was compared toassociation with a 31 nucleotide ARE construct. The effects of these two31-nucleotide RNA constructs on the availability of Mn²⁺ in low salt(˜12 mM Na⁺) solutions of 5 mM PIPES, pH 7.2, as measured in Example 3,are shown in FIG. 7. The effects of U₃₁ are represented by diamondswhile those of the 31-nt ARE construct are represented by triangles.This figure clearly demonstrates the base specific nature of metalbinding. Solid lines represent behavior predicted by a model wherein asingle binding site existed with K_(d) values of 38 (upper line) and 2.7μM (lower line). Dashed line (a) represents behavior predicted for amodel where the ARE construct bound a single metal ion with highaffinity.

This system was used to investigate the effects of added electrolytes onmetal binding. While added sodium chloride had little effect on Mn²⁺binding by the 31-nt ARE, addition of 100-mM Na⁺ resulted in negation ofthe effects of the U₃₁ construct. This result shows that Na⁺ competeseffectively with Mn²⁺ for association with the U₃₁ construct but notwith the ARE construct. The differences between the behaviors of U₃₁ andthe ARE construct indicate that the ARE construct has specific bindingsites for divalent cations, such as Mn²⁺. In the higher salt solution,50 μM RNA had about half the effect on magnetic relaxation than it didin the lower salt solution (data not shown).

Example 10 Titration of Mn²⁺ with A-Site RNA under Low Salt Conditions

In solutions containing 5 mM each of the sodium salts of MeOPH and PIPESbuffer (pH 7.2), the concentration of sodium ion is calculated to be˜12.5 mM. Addition of a 27-nt A-site construct to a solution of Mn²⁺under these conditions caused the uptake of multiple Mn²⁺ ions permolecule of RNA, as measured as in Example 3. The data are presented inFIG. 8. The behavior predicted for tight binding in a 1:1 stoichiometryis presented as dashed line (a). The series of unbroken curvesillustrates the behavior predicted by binding different numbers of metalions at assumed values of K_(d). In this model, all sites were assumedto have the same K_(d) and behave independently of whether other siteswere occupied. The lowest curve represents binding of four Mn²⁺ ionswith a K_(d) of 4.6 μM. The highest curve represents binding of 8 Mn²⁺ions with a K_(d) of 32 μM.

Example 11 Displacement of Metal Ions from the A-Site by Neomycin

FIG. 9 shows the effect of titration of a solution of a 27-nt A-site RNAconstruct (27 μM), complexed with Mn²⁺ (16 μM), with neomycin, measuredas in Example 3. Data in this figure show that metal ions were displacedby addition of the aminoglycoside drug, neomycin, as was suggested byMikkelsen et al (Mikkelsen, N. E. et al. Nature Struct. Biol. 2001,8:510–514). The data shown in FIG. 9 were most consistent with a modelwhere three neomycin molecules were bound to each RNA molecule withsubsequent release of Mn²⁺. Release of Mn²⁺ resulted in an increase inthe relaxation rate of the probe ion phosphorus nucleus.

Example 12 The Effects of Electrolytes on Magnetic Relaxation inSolutions of Mn²⁺ and RNA

The effect of K⁺ ion concentration on relaxation enhancement is shown inFIG. 10. While the addition of KCl solution resulted in an initialincrease in relaxation enhancement, measured as in Example 3, after theconcentration of monovalent ions exceeds about 40 mM, addition of moreKCl had little effect. This apparent saturation effect could have one oftwo possible origins: the monovalent ions were only able to displacesome percentage of the divalent ions from their binding sites, or atsufficiently high concentrations, monovalent ions associated with theRNA, neutralizing its negative charge. If the latter occurs, thenrelaxation of the phosphorous nucleus by the coordinated Mn²⁺ becomespossible.

Example 13 Determining the Effects of Potassium Ion on the Shapes ofTitration Curves

The effects of 140 mM K⁺ on the curves obtained when solutions of Mn²⁺are titrated with the 27-nt A-site construct are presented in FIG. 11.In contrast to the behavior observed in low salt, titrations in 140 mMK⁺ did not result in complete loss of relaxation enhancement, measuredas in Example 3. A comparison of titrations run in 3 and 6 μM Mn²⁺solutions shows that impurities introduced with the KCl or the bufferwere not responsible for the residual relaxation enhancement. Theseresults were most consistent with a model where association ofmonovalent ions with the A-site RNA resulted in a significant decreasein charge density, and as a result, the probe ion was able to contactthe Mn²⁺ while bound to the nucleic acid.

Example 14 Determining the Number of High Affinity Metal BindingPositions on RNA

Under suitable conditions, it will be possible to determine the numberof metal ions that are tightly bound by a given nucleic acid. Titrationof a solution of the nucleic acid into a solution containing theparamagnetic metal ion and the probe ion will result in a decrease inthe availability of the metal with the result that it will decrease therate of T₂ relaxation of the probe nuclei, measured as in Example 3. Thechange in free metal concentration can be determined from the change inthe relaxation rate. The following relationship applies:1/T _(2,a)−1/T _(2,b) =k{[M] _(a) −[M] _(b)}where the subscripts a and b refer to solutions a and b, and k is aconstant. For Mn²⁺ solutions k is equal to the second order rateconstant for reaction with the probe ion, for MeOPH, this value is5.8×10⁶ M⁻¹s⁻¹. Plotting the concentration of added nucleic acid versusthe change in metal concentration will give a curve where the initialslope is equal to the stoichiometric ratio of metal ions bound pernucleic acid. To prevent interference from non-specific metal binding,these experiments may be performed at varying ionic strengths using aninert electrolyte such as ammonium acetate.

Example 15 Developing Structure Activity Relationships (SAR's) fromAffinity Data

Using affinity data, structure activity relationships are developed torefine drug leads by established methods (Hajduik, P. J., et al.Quarterly Rev. Biophys. 1999, 32:211–240; Moore, J. M. Current Opin.Biotech., 1999, 10:54–58).

Example 16 Displacement of Mn²⁺ from RNA

NMR spectra were recorded on either a Varian Mercury 200 MHz, or aBruker Avance DRX 500 MHz NMR spectrometer. Transverse relaxation rates(R₂=1/T₂) were measured indirectly by exploiting the strong scalarcoupling between the phosphorus and the phosphorus bound hydrogen(J_(HP)=630 Hz). The one dimensional pulse sequence is described in FIG.12.

RNA constructs were purchased from Dharmacon (Lafayette, Colo.) alreadygel purified and desalted. The samples were washed repeatedly, firstwith 1 M NaCl, and then with H₂O, using a centricon filtration device.Samples were subsequently lyophylized and reconstituted in D₂O Prior touse. Neomycin b was purchased from Sigma (St. Louis, Mo.) and was usedwithout further purification. The K+ salt of MeOPH was prepared byhydrolysis of (MeO)₂P(H)O (Aldrich, St. Louis, Mo.) with KOH solution.The reaction was monitored by pH, and was evaporated to dryness invacuo. Similarly, K₂HPO₃ was prepared by neutralization of phosphorusacid (Aldrich) with KOH. Samples were pure by ¹H and ³¹P NMR and wereused without further purification. The RSG1.2 Peptide (Tan, R.; Frankel,A. D., Biochemistry 1994, 33: 14579–14585) was prepared by Synpep, Inc(Dublin, Calif.), with a TAMRA label on the N-terminus. All NMR studieswere performed in D₂O using PIPES as a non-complexing ionic buffer (Yu,Q.; Kandegedara, A.; Xu, Y.; Rorabacher, D. B., Ana.l Biochem. 1997,253: 50–56). The buffer was prepared as the K⁺ salt by neutralization ofthe free acid with KOH.

Mn²⁺ binding by RNA constructs, and its displacement by either K⁺ orMg²⁺. Samples containing MeOPH (5 mM) and PIPES buffer (5 mM, pH=7.2),and 3 μM Mn²⁺ were titrated with each RNA. The ³¹P transverse relaxationrates (R₂=1/T₂) of the probe in the resulting solutions were measured.Plots of R₂ versus [RNA] were prepared. The effects of [K⁺] and [Mg²⁺]on Mn²⁺ binding were determined by titrating similar solutionscontaining buffer, MeOPH, Mn²⁺ and one of the RNAs (10 to 30 μM) withsolutions of either KCl or MgCl₂, and monitoring the relaxation rate ofthe probe as a function of cation concentration. The effects of [K⁺]were studied over the range of 12.5 to 150 mM (intercellular K⁺ isreportedly 140 mM). Mn²⁺ displacements from each of the hairpins byMg²⁺, neomycin b, and RSG1.2 were studied in the absence of added KCland with sufficient KCl added to bring the total [K⁺] to 100 mM. Thecompounds employed in this example are shown in FIG. 13.

Displacement of Mn²⁺ from RNA by RSG1.2, or neomycin b: Binding ofRSG1.2 and neomycin b to each of the hairpins was studied at [RNA]between 20 and 30 μM, and again at [RNA]˜3 μM. The higher concentrationstudies were performed in a manner identical to that described above forthe Mg²⁺ exchange reactions. For the low concentration studies, HPO₃ ²⁻(20 mM) was used as the probe ion. Reactions were run in 10 mM PIPESbuffer, with 45 mM KCl and [Mn²⁺] between 50 and 150 nM. The pH of thesamples was maintained at 7.8 in these experiments. Studies utilizingHPO₃ ²⁻ are impractical at lower pH where the probe undergoes dynamicproton exchange (pK_(A)=6.0) leading to significant shortening of thediamagnetic ³¹P T₂.

Fluorescence polarization studies of RSG1.2/RNA binding: Samplescontaining the N-terminally TAMRA labeled peptide, RSG1.2 (3 nM) andeither RRE1 or RRE2 (concentrations ranging from 0.1 to 200 nM) wereprepared in Reaction Buffer (10 mM Hepes-KOH pH 7.5; 150 mM KCl; 5 mMMgCl₂; 1 mM DTT; 1% glycerol; 50 μg/ml BSA; 100 μg/ml Hen-Egg Lysozyme).Each sample was irradiated at 530 nM and the polarization of thefluorescent emission at 580 nM was recorded using a LJL Analyst(Molecular Devices Corp. Sunnyvale, Calif.). Measurements repeated at 1,15, and 30 min showed no evidence of change with time.

Measurable Mn²⁺ binding occurs under a useful range of [RNA]. Thesensitivities of the ³¹P T₂ NMR relaxation rates of phospite (HPO₃ ²⁻)and methyl phosphite (MeOPH) to Mn²⁺ make it possible to measure [Mn²⁺ ]accurately at sub-micromolar levels (FIG. 14) (Summers et al. Inorg.Chem., 2001, 40: 6547–6554). Titration of each of the four RNAs intosolutions containing Mn²⁺ (3 μM), MeOPH (5 mM), and PIPES buffer (5 mM,pH 7.2) caused a diminution of the MeOPH ³¹P transverse relaxation rate(R2), indicating that the RNAs sequestered the Mn²⁺. The effect of theA-site construct on free [Mn²⁺] (FIG. 15) is typical. In 12 mM K⁺, thethree hairpins bound half the available Mn²⁺ at much lowerconcentrations (3 to 7 μM) than was required for U31 (˜50 μM). At 100μM, the affinity of each hairpin RNA was sufficient that relaxationenhancement of the probe by 3 μM Mn²⁺ was undetectable. Since efficientrelaxation enhancement requires inner sphere contact between MeOPH andthe Mn²⁺ ion (Summers et al. Inorg. Chem., 2001, 40: 6547–6554), thisresult indicates that the anionic probe does not contact the RNAcomplexed metal ion. Thus, the ³¹P T₂ relaxation enhancement of MeOPHcan be used to measure the concentration of free Mn²⁺ ion withoutinterference by RNA complexed Mn²⁺.

While these experiments provide an accurate measure of the concentrationof free Mn²⁺, the certainty of Mn²⁺ dissociation constants (K_(d,Mn))derived from such data is limited by the uncertainty in the number ofdiscreet binding sites. The line in FIG. 15 was produced assuming thatthe RNA has 7 non-interacting binding sites, each with K_(d,Mn)=25 μM.We note, however, that the data fit just as well to a model with 8 ormore binding sites having higher K_(d,Mn) values. A Scatchard analysisof binding data would require knowledge of RNA saturation which is notavailable from this technique. Additional uncertainty is introduced bythe potential presence of trace impurities (diamagnetic metal ions,and/or high affinity chelating agents such as EDTA) in the RNA samples.We feel that our K⁺, Mg²⁺, neomcyin b, and RSG1.2 samples are not nearlyas prone to contamination as the RNA samples and have greater confidencein values derived from experiments wherein the concentration of thecation is varied. The 25 μM value is well within the range ofdissociation constants published for Mn²⁺/RNA interactions: in 0.1 MNaCl, the hammerhead ribozyme reportedly binds four Mn²⁺ ions withK_(d,Mn)˜4 μM and another five with K_(d,Mn)˜460 μM (Hoogstraten et al.J. Am. Chem. Soc., 2002, 124: 834–842).

As observed in earlier studies, Mn²⁺/RNA binding was strongly influencedby the concentration of monovalent ions. At [K⁺]<20 mM, addition of KClsolution results in increased relaxation enhancement, above 40 mM,however, the K⁺ association becomes saturated and further addition ofKCl had little effect. The effect of [K⁺] on Mn²⁺ binding by the A-siteconstruct is presented in FIG. 16. The effects of K⁺ on Mn²⁺ binding bythe RNAs were sequence dependent. While 30 mM K⁺ displaced all the Mn²⁺from U₃₁, 150 mM K⁺ did not displace all the Mn²⁺ from any of the threehairpins. This result indicates that the hairpins contain discretebinding sites that are selective for divalent metals and that U₃₁ doesnot. We considered the possibility that high [K⁺] might allow contactbetween the probe ion and the RNA bound Mn²⁺. This hypothesis wasinconsistent with the results of experiments comparing the relaxationenhancements of the two probes; MeOPH and HPO₃ ²⁻. The two probes (whichhave different electronic charges) should differ in sensitivity to theelectronic environment of the Mn²⁺. This reasoning was borne out byexperiments comparing the relative sensitivities of MeOPH and HPO₃ ²⁻toward relaxation by Mn²⁺ in the presence and absence of EDTA.Relaxation of the di-anionic probe HPO₃ ²⁻ by the anionic metal complex(MnEDTA²⁻) was found to be significantly slower than that of themono-anionic probe (MeOPH) (Results not shown). While addition of K⁺ toMn²⁺ containing solutions of RRE1, caused an increase in the relaxationrates of both probes, the ratio of the two remained equal to thatobserved in the absence of the RNA. Since the electronic chargeenvironment surrounding the complexed Mn²⁺ should be significantlydifferent than that of the free ion, this result indicates that additionof K⁺ causes an increase in the concentration of free Mn²⁺, which is thesole species responsible for probe nucleus relaxation in theseexperiments. The decrease in Mn²⁺ affinity is likely to stem from acombination of two effects: First, K⁺ is able to compete with Mn²⁺ fornon-specific electrostatic interactions, and second, neutralization ofthe anionic charge causes a decrease in the affinity of specificdivalent metal sites.

Mn²⁺ is displaced from RNA by Mg²⁺ in competition experiments. Todetermine whether Mn²⁺ was bound at sites on the RNA molecules that bindMg²⁺ in vivo, we titrated solutions containing Mn²⁺ and the RNAs withMgCl₂ solution and monitored the effect using the PhoRE technique. Theeffect of Mg²⁺ on Mn²⁺ binding by U₃₁ was that expected for competitionbetween two identical cations for association with an indiscriminatepoly-anion; the probe R₂ increased with [Mg²⁺] until the concentrationsof the two ions were similar. After the [Mg²⁺] had exceeded about twicethe [Mn²⁺], addition of more Mg²⁺ had no measurable effect.

Mn²⁺ bound by the hairpins was displaced by Mg²⁺ in a manner moreconsistent with the metals being bound in a well defined complex. Theeffects of [Mg²⁺] on Mn²⁺ binding by RRE1 at two different [K⁺] arepresented in FIG. 17. Titration curves were interpreted in terms of amodel that assumes that [Mn²⁺]<<K_(d,Mn) and that the Mg²⁺ bindingequilibrium is governed by the Hill equation. Under these conditions theeffect of [Mg²⁺] on the observed relaxation rate (R_(2,obs)) is governedby Eq (1):(Q−Q ₀)/Q ₀=(1/K _(d,Mg))[Mg] ^(n)  (1)where Q=(R_(2,obs)−R_(2,dia))/(R_(2,max)−R_(2,obs)), R_(2,dia) andR_(2,max) represent the values of R₂ in diamagnetic solutions and themaximum value observed, respectively. The term Q₀ (defined asQ₀=K_(d,Mn)/RNA) arises from the competitive nature of the experiment,and represents the value of Q observed in the absence of added Mg²⁺. Thepresence of Q₀ in Eq (1) introduces a minor complication in theinterpretation of the data which can be illustrated by considering thedata presented in FIG. 17. While the concentrations of Mg²⁺ required torelease half the bound Mn²⁺ were similar for the two experiments (300and 600 μM), Q₀ values were significantly different. As a resultK_(d,Mg) determined in 25 mM K⁺ was considerably lower than in 100 mM K⁺(64 versus 360 μM, Table 1).

TABLE 1 Equilibrium coefficients for Mg²⁺ binding by RNA constructs.K_(d,Mg) (μM) (Hill Coefficient) RNA 25 mM K⁺ 100 mM K⁺ U₃₁ none A-site55 (0.8) 154 (1.0) RRE1 64 (2.8) 360 (1.5) RRE2 344 (1.0)

Using the analysis described above, values of K_(d,Mg) and n weredetermined for A-site and RRE1 at low [K⁺] and at saturating [K⁺] (Table1). Like RRE1, the A-site also showed a marked decrease in Mg²⁺ affinityat the higher [K⁺]. The decrease in Mg²⁺ affinity at the higher [K⁺]mirrors the decrease in Mn²⁺ affinity noted above. All the values inTable 1 are well within the range of those found in the literature;values of K_(d,Mg) ranging from 1 μM to 2 mM have been reported fortransfer RNAs alone (Schimmel et al. Ann Rev. Biophys. Bioeng., 1980, 9:181–221).

Neomycin b reacted with each of the hairpins, causing a release of Mn²⁺.The Mn²⁺ release was proportional to the added neomycin until the RNAwas saturated. After saturation, continued addition of neomycin had noeffect on MeOPH relaxation (FIG. 18). The ratio of neomycin to RNA atthe saturation point was dependant on the RNA sequence, [K⁺], and [RNA].At [RNA]=2.7 μM (100 mM K⁺), the A-site construct bound a singleequivalent of neomycin (FIG. 18). At [RNA]=27 μM, however, the sameconstruct bound 3 equivalents (data not shown). Both RRE1 and RRE2constructs bound multiple equivalents of neomycin under each conditionstudied. Thus, of the three hairpins, only the A-site construct boundneomycin in a 1:1 complex, and only at RNA<3 μM. The low selectivity forneomycin binding should not be surprising since the antibiotic is knownto bind a wide variety of RNAs with varying affinities (Hendrix et al.J. Am. Chem. Soc., 1997, 119: 3641–3648; Sannes-Lowery et al. Anal.Biochem., 2000, 280: 264–271. ). We note that another RRE1 model boundthree equivalents of neomycin b (Harada et al. Nature, 1996, 380:175–179).

Binding RSG1.2 Peptide to RNA constructs was also strongly dependent onRNA sequence, [RNA] and [K⁺]. At <3 μM RNA, 100 mM K⁺, the RRE1construct bound a single equivalent of RSG1.2 (FIG. 19). Under theseconditions neither the RRE2 construct, nor the A-site bound the peptide.At higher RNA (>20 μM), each RRE construct bound multiple equivalents ofRSG1.2, releasing Mn²⁺, resulting in precipitation of the RNA/peptidecomplex. The results of our PhoRE studies of RSG1.2 /RNA binding areconsistent with those of our fluorescence polarization studies of thissystem. The effects of RRE1 and RRE2 on fluorescence polarization ofTAMRA labeled RSG1.2 are presented in FIG. 20. Low levels of RRE1 causean increase in polarization consistent with complexation of the peptideand an increase in effective mass. A similar level of polarizationrequires a fifty fold greater concentration of RRE2, indicating a weakerinteraction between the peptide and this RNA.

Our results demonstrate that PhoRE represents a viable method forstudying the interactions of metal ions, small molecules, and peptideswith nucleic acids. We describe evidence for discreet divalent metalbinding sites on three hairpin RNAs. We found that Mg²⁺ is able tocompete with Mn²⁺ at physiological concentrations, indicating that smallmolecules, peptides, or proteins that displaces Mn²⁺ from an RNA shouldbind in vivo by displacing Mg²⁺.

Other Embodiments

Modifications and variations of the described methods of the inventionwill be apparent to those skilled in the art without departing from thescope and spirit of the invention. Although the invention has beendescribed in connection with specific desirable embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention, which are obvious tothose skilled in the field of chemistry or related fields, are intendedto be within the scope of the invention.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually to be incorporated by reference.

Other embodiments are in the claims.

1. A method of detecting the binding of a species to a nucleic acid,said method comprising the steps of: (a) measuring a magnetic relaxationproperty of a probe in a first solution using nuclear magnetic resonancespectroscopy, wherein said first solution comprises said nucleic acid, afirst concentration of said species, a paramagnetic metal ion, and saidprobe; (b) measuring the magnetic relaxation property of said probe in asecond solution using nuclear magnetic resonance spectroscopy, whereinsaid second solution comprises said nucleic acid, a second concentrationof said species, a paramagnetic metal ion, and said probe; and (c)comparing the relaxation property measured in step (a) with therelaxation property measured in step (b), wherein a difference in saidrelaxation properties indicates the binding of said species to saidnucleic acid.
 2. The method of claim 1, wherein, in step (c), saidmagnetic relaxation properties of said probe are correlated with saidfirst and second concentrations of said species, thereby quantifying thebinding of said species to said nucleic acid.
 3. The method of claim 1,wherein said first concentration is 0.0 μM.
 4. The method of claim 1,wherein said species is a divalent metal ion.
 5. The method of claim 4,wherein said metal ion is Mg²⁺.
 6. The method of claim 1, wherein saidspecies is a candidate therapeutic agent.
 7. The method of claim 6,wherein said candidate therapeutic agent is selected from a molecularlibrary.
 8. The method of claim 1, wherein said species is selected fromthe group consisting of a protein, a peptide, and a fragment of aprotein.
 9. The method of claim 1, wherein said paramagnetic metal ionis selected from the group consisting of Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺,or a lanthanide ion.
 10. The method of claim 1, wherein saidparamagnetic metal ion is bound to said nucleic acid.
 11. The method ofclaim 1, wherein said probe comprises an X—H bond, wherein X is anNMR-active nucleus.
 12. The method of claim 11, wherein X is ³¹P. 13.The method of claim 12, wherein said probe is methylphosphite,ethylphosphite, or ethylphosphonite.
 14. The method of claim 11, whereinsaid magnetic relaxation property of X is indirectly detected using Xedited, ¹H detected NMR spectroscopy.
 15. The method of claim 14,wherein said measuring of said magnetic relaxation properties of Xproceeds by the steps of: (i) using a pulse sequence to transfercoherent magnetization originating on the ¹H nucleus to the X nucleus byInsensitive Nuclei Enhanced by Polarization Transfer (INEPT) techniquesprior to a T₂ delay; (ii) providing a T₂ delay; and (iii) transferringthe remaining coherence back to the ¹H nucleus for detection using areverse INEPT series of pulses.
 16. The method of claim 1, wherein saidmagnetic relaxation property is the T₂ relaxation of a nucleus of saidprobe.
 17. The method of claim 1, wherein said nucleic acid comprisesRNA.
 18. The method of claim 1, wherein said nucleic acid comprises DNA.19. The method of claim 1, wherein said nucleic acid is double-stranded.20. A method of detecting the binding of a paramagnetic metal ion to anucleic acid, said method comprising the steps of: (a) measuring amagnetic relaxation property of a probe in a first solution usingnuclear magnetic resonance spectroscopy, wherein said first solutioncomprises a first concentration of said paramagnetic metal ion, saidnucleic acid, and said probe; (b) measuring the magnetic relaxationproperty of said probe in a second solution using nuclear magneticresonance spectroscopy, wherein said second solution comprises a secondconcentration of said paramagnetic metal ion, said nucleic acid, andsaid probe; and (c) comparing the relaxation property measured in step(a) with the relaxation property measured in step (b), wherein adifference in said relaxation properties indicates the binding of saidparamagnetic metal ion to said nucleic acid.
 21. The method of claim 20,wherein, in step (c), said magnetic relaxation properties of said probeare correlated with said first and second concentrations of said nucleicacid, thereby quantifying the binding of said paramagnetic metal ion tosaid nucleic acid.
 22. The method of claim 20, wherein said firstconcentration is 0.0 μM.
 23. The method of claim 20, wherein saidparamagnetic metal ion is selected from the group consisting of Mn²⁺,Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, or a lanthanide ion.
 24. The method of claim 1,wherein said probe comprises an X—H bond, wherein X is an NMR-activenucleus.
 25. The method of claim 18, wherein X is ³¹P.
 26. The method ofclaim 25, wherein said probe is methylphosphite, ethylphosphite, orethylphosphonite.
 27. A method of determining the effect of a firstspecies on the binding of a second species to a nucleic acid, saidmethod comprising the steps of: (a) measuring a magnetic relaxationproperty of a probe in a first solution using nuclear magnetic resonancespectroscopy, wherein said first solution comprises a firstconcentration of said first species, said nucleic acid, said secondspecies that binds to said nucleic acid, a paramagnetic metal ion, and aprobe; (b) measuring the magnetic relaxation property of said probe in asecond solution using nuclear magnetic resonance spectroscopy, whereinsaid second solution comprises a second concentration of said firstspecies, said nucleic acid, said second species, said paramagnetic metalion, and said probe; and (c) comparing the relaxation property measuredin step (a) with the relaxation property measured in step (b), wherein adifference in said relaxation properties indicates the inhibition of thebinding of said second species to said nucleic acid.
 28. The method ofclaim 27, wherein, in step (c), said magnetic relaxation properties ofsaid probe are correlated with said first and second concentrations ofsaid first species, thereby quantifying the effect of said first specieson the binding of said second species to said nucleic acid.
 29. Themethod of claim 27, wherein said first concentration is 0.0 μM.
 30. Themethod of claim 27, wherein one of said first species and said secondspecies is a divalent metal ion.
 31. The method of claim 30, whereinsaid metal ion is Mg²⁺.
 32. The method of claim 27, wherein said firstspecies or said second species is a candidate therapeutic agent.
 33. Themethod of claim 32, wherein candidate therapeutic agent is selected froma molecular library.
 34. The method of claim 27, wherein said first orsaid second species is selected from the group consisting of a protein,a peptide, and a fragment of a protein.
 35. The method of claim 27,wherein said paramagnetic metal ion is selected from the groupconsisting of Mn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, or a lanthanide ion.
 36. Themethod of claim 27, wherein said paramagnetic metal ion is bound to saidnucleic acid.
 37. The method of claim 27, wherein said probe comprisesan X—H bond, wherein X is an NMR-active nucleus.
 38. The method of claim27, wherein X is ³¹P.
 39. The method of claim 38, wherein said probe ismethylphosphite, ethylphosphite, or ethylphosphonite.
 40. A method ofdetecting the availability of a paramagnetic metal ion in a solutioncomprising a nucleic acid, said method comprising the steps of: (a)providing a solution comprising said nucleic acid, a probe, and saidparamagnetic metal ion and (b) measuring a magnetic relaxation propertyof said probe in said solution using nuclear magnetic resonancespectroscopy, wherein the magnitude of said magnetic property isindicative of the availability of said paramagnetic ion in saidsolution.
 41. The method of claim 40, wherein said paramagnetic metalion is bound to said nucleic acid.